Immunoassays
Immunoassays have found widespread application in the field of clinical diagnostics for the detection and measurement of drugs, vitamins, hormones, proteins, metabolites, microorganisms, and other substances of interest (analytes) in biological and non-biological fluids. Typically, these analytes occur in micromolar (10.sup.-6 M) or less concentration.
Immunoassays generally incorporate antibodies and antigens as reactants, at least one of which is labeled with a signal-producing compound (e.g., radioisotope, fluorophore, enzyme, etc.). Following mixture with the sample and incubation, specific antibody/antigen reactions occur (specific binding). The reaction mixture is subsequently analyzed to detect free and specifically bound labeled reactant, enabling a measurement of the analyte in the sample.
Immunoassays can be divided into two general categories, homogeneous and heterogeneous. In a homogeneous immunoassay, the signal emitted by the specifically bound labeled reactant is different from the signal emitted by the free labeled reactant. Hence, bound and free can be distinguished without physical separation.
The archetypal homogeneous immunoassay is the enzyme-multiplied immunoassay technique (EMIT) which is disclosed in U.S. Pat. No. 3,817,837. In this technology, analyte present in patient sample and analyte/enzyme conjugate compete for a limited amount of anti-analyte antibody. Specific binding of antibody to the conjugate modulates its enzymatic activity; hence, the amount of enzyme activity is proportional to the amount of analyte in the sample.
Homogeneous immunoassays have the advantages of being rapid, easy to perform, and readily amenable to automation. Their principal disadvantages are that they are relatively prone to interferences, are generally limited to low molecular weight analytes, and are generally limited in sensitivity to approximately 10.sup.-9 M.
In a heterogeneous immunoassay, the signal emitted by the bound labeled reactant is indistinguishable from the signal emitted by the free labeled reactant; therefore, a separation step is required to distinguish between the two. Typical heterogeneous immunoassays include the radioimmunoassay (RIA) and the enzyme-linked immunosorbent assay (ELISA).
In the RIA, radiolabeled analyte and analyte present in patient sample compete for a limited amount of immobilized (solid-phase) anti-analyte antibody. The solid phase is washed to remove unbound, labeled analyte, and either the bound or the free fraction is analyzed for the presence of labeled reactant. ELISA assays are performed analogously. In the latter case though, the signal is an enzyme instead of a radioisotope. Heterogeneous immunoassays typically employ at least one reactant immobilized on a solid phase. Solids used to immobilize reactants in immunoassays have included controlled pore glass and preformed polymers, such as polyvinyls, polyacrylamides, polydextrans, and polystyrenes. Numerous separation methods are known in the art and have been used in heterogeneous immunoassays. These include centrifugation, micro-filtration, affinity chromatography, and gel-permeation chromatography. Since the kinetics of reaction between an immobilized antibody (or antigen) and its binding site tend to be slower than the kinetics of the same reaction occurring in solution, long incubation times are frequently required. When the multiple wash steps often needed are considered, it can be appreciated that heterogeneous assays tend to be time-consuming and labor-intensive. However, they are in general more sensitive than homogeneous assays and less prone to interferences, since interfering substances can be removed in the wash step(s).
Recently, a solid-phase immunoassay has been disclosed which is directed toward the concentration of signal to a very small surface area (EP 124,050; Jolley). Within this method, an analyte is reacted with an immunoreactant immobilized on water-insoluble particles (latex beads) in a substantially suspended state, and thereafter concentrated by microfiltration to a volume substantially less than the volume of the original sample. However, in addition to suffering from slow reaction kinetics, the use of latex beads as the solid phase contributes to high nonspecific binding. Further, since the signal bound to the latex beads collected on the filtration membrane is not physically bound to the membrane, this method is not suitable for a dipstick assay format, making it even less attractive for use in an efficient clinical setting.
Even more recently, an immunoassay has been developed in which membrane-linked antibody is used to immobilize an analyte, and immune reactions utilizing enzyme-linked antibody are carried out on a solid phase. Although this provides an improved method for concentrating signal within a small surface area, it still suffers from numerous disadvantages. These include slow reaction kinetics (because of the solid-liquid immune reaction), chemical modification of the membranes to covalently link the antibody to the membrane surface, high nonspecific binding of the membranes (generally nylon and glass fiber), a multiplicity of steps to carry out the assay, and the need for multiple high affinity antibodies. These disadvantages make this method practically unsuitable for detecting a wide variety of analytes within a clinical setting.
There is a need in the art, then, for an immunoassay which is highly sensitive, has fast-reaction kinetics, and which is readily amenable for use in efficiently detecting the presence of a variety of analytes within a clinical setting. The present invention fulfills this need and further provides other related advantages.